Concerns that variola (smallpox) virus might be used as a biological weapon have led to the recommendation of widespread vaccination with vaccinia virus (VACV) (Henderson, D. A. 1999 Science 283:1279-1282). While vaccination is generally safe and effective for prevention of smallpox, it is well documented that various adverse reactions in individuals have been caused by vaccination with existing licensed vaccines (Fulginiti, V. A. et al. 2003 Clin Infect Dis 37:251-271). Vaccinia immune globulin (VIG) prepared from vaccinated humans has historically been used to treat adverse reactions arising from VACV immunization (Kempe, C. H. 1960 Pediatrics 26:176-189; Feery, B. J. (1976) Vox Sang 31:68-76; Hopkins, R. J. et al. 2004 Clin Infect Dis 39:759-766; Hopkins, R. J. & Lane, J. M. 2004 Clin Infect Dis 39:819-826) and to date, VIG is still the only recommended treatment (Hopkins, R. J. et al. 2004 Clin Infect Dis 39:759-766; Hopkins, R. J. & Lane, J. M. 2004 Clin Infect Dis 39:819-826). However, VIG lots may have different potencies and carry the potential to transmit other viral agents.
VACV is the prototype virus in the genus Orthopoxvirus, which includes variola virus, the causative agent of smallpox. There are two major forms of infectious VACV: intracellular mature virus (MV) and extracellular enveloped virus (EV). The majority of the MV remains within the cell until lysis, but some are wrapped in additional membranes and exocytosed as EV. Most EV remains attached to the outside of the plasma membrane and is responsible for direct cell-to-cell spread; however some are released into the medium and can cause comet-like satellite plaques (Blasco, R. & Moss, B. 1992 J Virol 66:4170-4179; Blasco, R. et al. 1993 J Virol 67:3319-3325). The EV is important for virus dissemination in vivo as well as in cultured cells (Payne, L. G. 1980 J Gen Virol 50:89-100; Smith, G. L. & Vanderplasschen, A. 1998 Adv Exp Med Biol 440:395-414). Because an EV is essentially an MV enclosed by an additional membrane, the two forms of VACV have different outer proteins and bind to cells differently (Vanderplasschen, A. et al. 1998 J Gen Virol 79:877-887), though ultimately only the proteins of the MV membrane mediate membrane fusion (Moss, B. 2005 Virology 344:48-54). B5 is one of five known EV-specific proteins and is highly conserved among different strains of VACV as well as in other orthopoxviruses (Engelstad, M. et al 1992 Virology 188:801-810; Isaacs, S, N. et al. 1992 J Virol 66:7217-7224). B5 is a 42-kDa glycosylated type I membrane protein with a large ectodomain composed of four small domains that are similar to short consensus repeat (SCR) domains of complement regulatory protein (Engelstad, M. et al 1992 Virology 188:801-810; Isaacs, S, N. et al. 1992 J Virol 66:7217-7224) although no complement regulatory activity has been demonstrated. B5 is required for efficient envelopment of MV, as well as for actin tail formation, normal plaque size and virulence (Engelstad, M. & Smith, G. L. 1993 Virology 194:627-637; Sanderson, C. M. et al. 1998 J Gen Virol 79:1415-1425; Wolffe, E. J. et al. 1993 J Virol 67:4732-4741).
The B5 protein is an important target for neutralizing antibodies: antisera to B5 can neutralize EV in a plaque reduction assay and inhibit “comet formation”, the in vitro manifestation of cell-to-cell spread of EV (Engelstad, M. et al 1992 Virology 188:801-810, Galmiche, M. C. et al. 1999 Virology 254:71-80; Law, M. & Smith, G. L. 2001 Virology 280:132-142; Aldaz-Carroll et al. 2005 J Virol 79:6260-6271). Recent studies showed that anti-B5 in VIG was responsible for most of the neutralizing activity against EV as measured by a plaque reduction assay (Bell, E. et al. 2004 Virology 325:425-431). To date, rat and mouse anti-B5 neutralizing MAbs have been reported (Aldaz-Carroll et al. 2005 J Virol 79:6260-6271; Schmelz, M. et al. 1994 J Virol 68:130-147) and the epitopes recognized by mouse MAbs have been mapped to the border of SCR1-SCR2 and/or the stalk of B5 (Aldaz-Carroll et al. 2005 J Virol 79:6260-6271). In addition, a rat monoclonal antibody (MAb) to B5 provided protection in a VACV mouse challenge model (Lustig, S. et al. 2005 J Virol 79:13454-13462).